SPECIFIC AIMS Antibiotic resistance among common bacterial pathogens is a serious public health problem as it compromises our ability to treat infectious disease. The resistance problem is compounded by the relative lack of discovery of new antibiotics, especially those with novel mechanisms of action. New antibiotics are critically needed as resistance to recently developed antibiotics is growing. The goal of this project is to develop our triazolononactate antibiotics, a novel structural class of compound, as broad spectrum agents active against Gram positive pathogens such as methicillin-resistant Staphylococcus aureus. Based on the success of our Phase I STTR project we will seek to improve both potency and selectivity against important pathogens, identify and validate the molecular target through which our antibiotics exert their bactericidal activity and, after in vitro and in vivo DMPK analysis, demonstrate efficacy in a mouse model of infection. The data obtained will be used to demonstrate the potential of the compound class, the goal of which will be to partner/out- license with others to complete the enabling work for the filing of an IND application. Specific Aim 1 Lead compound improvement and evaluation. We have demonstrated that we can synthesize diverse triazolononactate derivatives and through preliminary SAR studies we have improved the potency (MIC) against clinically relevant pathogens to around MIC values of 1-4 g/mL. While encouraged by these studies we seek to further improve potency and selectivity through additional synthesis and SAR studies. Specific Aim 2 Target identification and validation. Drug development has been greatly facilitated where knowledge of the drug target can be used to understand SAR. We will determine the discrete molecular target at which our triazolononactate antibacterial agents act. We will use a combination of approaches photoaffinity labeling of the triazolononactate target and the genetic analysis of S. aureus triazolononactate resistance mutants. Specific Aim 3 Compound evaluation procedures. The third aim of this project encompasses all the required analytical methods for compound evaluation, ranging from initial measures of potency through to eventual demonstration of efficacy in a mouse neutropenic thigh burden model. The analytical work is done in conjunction with the SAR studies of specific aim 1 so that iterative cycles of design, synthesis and evaluation will lead to a compound optimized for potency, selectivity, lack of off-target pharmacology, druggability and in vivo efficacy.